Method for preparing 1,3-propanediol by a recombinant micro-organism in the absence of coenzyme b12 or one of its precursors

Abstract

The invention concerns a method for preparing 1,3-propanediol from a carbon-containing substance, said method comprising a step which consists in culturing a recombinant micro-organism not producing coenzyme B12 in the absence of coenzyme B12 or one of its precursors. The invention also concerns a nucleic acid coding for a glycerol dehydratase whereof the catalytic activity is independent of the presence of coenzyme B12 or one of its precursors and a nucleic acid coding for a 1,3-propanol dehydrogenase intervening in the synthesis of 1,3-propanediol. The invention further concerns recombinant vectors and host cells comprising said nucleic acids and the polypeptides coded by the latter.

1. A recombinant nucleic acid coding for at least one subunit of a glycerol dehydratase, wherein the catalytic activity of the glycerol dehydratase is not dependent on coenzyme B12 or one of its precursors, wherein the nucleic acid encodes a glycerol dehydratase and comprises a polynucleotide region comprising at least 90% nucleotide identity with the nucleic acid sequences of SEQ ID NO. 1 or SEQ ID NO. 2, or a polynucleotide with a nucleotide sequence that is fully complementary to a polynucleotide region comprising at least 90% nucleotide identity with the nucleic acid sequences of SEQ ID NO. 1 or SEQ ID NO. 2. 
     
     
       2. The recombinant nucleic acid of  claim 1 , wherein the nucleic acid further encodes for two sub-units of the glycerol dehydratase. 
     
     
       3. A recombinant nucleic acid coding for a glycerol dehydratase, wherein the catalytic activity of the glycerol dehydratase is not dependent on coenzyme B12 or one of its precursors, wherein the nucleic acid comprises:
 (a) a first polynucleotide region having at least 90% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 1; and 
 (b) a second polynucleotide region having at least 90% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 2. 
 
     
     
       4. The recombinant nucleic acid of  claim 3  further comprising a third polynucleotide region having at least 90% nucleotide identity with SEQ ID NO 4. 
     
     
       5. The recombinant nucleic acid of  claim 4 , wherein SEQ ID NO. 1 and SEQ ID NO. 2 are positioned 5′ to SEQ ID NO. 4. 
     
     
       6. The recombinant nucleic acid of  claim 4 , wherein the nucleic acid comprises at least 90% nucleotide identity with the nucleic acid sequence of SEQ ID NO. 5. 
     
     
       7. The recombinant nucleic acid of  claim 4  further comprising fourth polynucleotide region coding for a glycerol-3-phosphate dehydrogenase and a fifth polynucleotide region coding for a glycerol-3-phosphatase. 
     
     
       8. The recombinant nucleic acid of  claim 3 , wherein the nucleic acid further comprises a sequence with a transcription promoter function. 
     
     
       9. The recombinant nucleic acid of  claim 8 , wherein the promoter sequence comprises at least 80% nucleotide identity with SEQ ID NO. 3. 
     
     
       10. The recombinant nucleic acid of  claim 8 , wherein the promoter sequence comprises SEQ ID NO. 3. 
     
     
       11. A vector comprising the recombinant nucleic acid of  claim 1 . 
     
     
       12. The vector of  claim 11 , which is an expression vector. 
     
     
       13. The vector of  claim 11 , which is a cloning vector. 
     
     
       14. An isolated recombinant host cell comprising the recombinant nucleic acid of  claim 1 . 
     
     
       15. The host cell of  claim 14 , which is an  Escherichia coli  strain filed at the National Collection of Culture of Micro-organisms (NCCM) on Jun. 24, 1999 under the access No. I-2243. 
     
     
       16. The vector of  claim 11 , which is plasmid pSPD5. 
     
     
       17. A recombinant nucleic acid sequence with a bacterial promoter function comprising a polynucleotide region having at least 80% nucleotide identity with the sequence SEQ ID NO. 3, or a polynucleotide with a nucleotide sequence that is fully complementary to a polynucleotide region having at least 80% nucleotide identity with the sequence of SEQ ID NO. 3. 
     
     
       18. A process for making a polypeptide comprising at least one subunit of a glycerol dehydratase, wherein the catalytic activity of the glycerol dehydratase is not dependent on coenzyme B12, comprising:
 (a) preparation of an expression vector comprising a recombinant nucleic acid encoding a glycerol dehydratase having at least 90% amino acid identity with SEQ ID NO. 6 or SEQ ID NO. 7, a recombinant nucleic acid encoding a dimeric protein having glycerol dehydratase activity comprising a first polypeptide comprising at least 90% amino acid identity to SEQ ID NO. 6 and a second polypeptide comprising at least 90% amino acid identity to SEQ ID NO. 7, or a recombinant nucleic acid that has at least 90% nucleotide identity with SEQ ID NO. 4 and encodes a 1,3-propanediol dehydrogenase comprising an amino acid sequence of at least 90% amino acid identity to SEQ ID NO. 8; 
 (b) introduction of the expression vector into a host cell; 
 (c) culture of the host cell in a suitable medium; and 
 (d) recovery of the polypeptide produced from the host cell. 
 
     
     
       19. The process of  claim 18  further comprising purifying the polypeptide produced from the host cell. 
     
     
       20. The process of  claim 18 , wherein the polypeptide is recovered from the culture supernatant or the cell lysate. 
     
     
       21. The process of  claim 18 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 7, or a dimeric protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO. 6 and a second polypeptide comprising the amino acid sequence of SEQ ID NO. 7, or a polypeptide encoded by a recombinant nucleic acid comprising a first polynucleotide region comprising SEQ ID NO: 4 coding for a 1,3-propanediol dehydrogenase comprising the amino acid sequence of ID NO. 8.